Previously, we showed that mutations in the gene encoding connexin32 (Cx32) caused a demyelinating peripheral neuropathy called Charcot-Marie-Tooth disease (CMTX). Consistent with this finding, Schwann cells contain Cx32 and regulate its expression like a myelin-related gene. Thus, maintenance of myelin in the human peripheral nervous system requires connexin expression. However, oligodendrocytes also express and regulate Cx32 like a myelin gene and yet central abnormalities are rare in CMTX patients. Since one explanation for this discrepancy would be redundant expression of other connexins, we searched for connexins in myelinating glia. We found two novel connexins, Cx29 and Cx47. All three connexins can be found in oligodendrocytes and Schwann cells. Cx29 and Cx32, however, are present in non-overlapping subsets of spinal cord oligodendrocytes and,while they are both present in Schwann cells, their subcellular distributions are strikingly different. Single knockouts of either Cx32 or Cx47 myelinate relatively normally and have no functional deficits. In contrast, double knockouts develop severe central demyelination and die during the 6th postnatal week of life. Surprisingly, these animals display only subtle abnormalities in peripheral myelin. Together, our studies suggest that connexins are critical for both central and peripheral myelination but that different connexins may have different functions within myelinating glia. We propose to define the separate and interacting roles of connexins in myelination using a combination of immunocytochemistry, targeted gene ablation and functional analysis of connexin channel activity.